Molecular Cloning And Functional Characterization Of CpMYC2 And CpbbHLH13 Transcription Factors From Wintersweet(Chimonanthus Praecox)(L.)

Wintersweet（Chimonanthus praecox L.）an ornamental and economically important plant native to China is known for its beautiful flower coloration,strong fragrance as well as unique flower time which makes it highly desirable type of ornamental plant.Due to the less availability of basic genomics and transcriptomic data,our understanding of genomics,molecular biology and genetics of wintersweet is limited.At the full open stage,flowers of Ch.praecox emit abundant volatile compounds including monoterpenes and sesquiterpenes especially Linalool,β-ocimene and benzyl ester while recently a range of flavonols and anthocyanins has been detected from yellow to red flower petals at the full bloom stage.In this study,we selected two bHLH transcription factors named as CpMYC2 and CpbHLH13 from wintersweet H29（Huazhong 29）cDNA transcriptomic library which was constructed from five different growing stages of flower.The complete CDS sequence of both the genes was verified from the recently constructed genomics library of wintersweet H29.Sequencing analysis revealed that the full length open reading frame of CpMYC2 contains 1884 bp nucleotides and encodes a protein consists of 627 amino acids.While CpbHLH13 contains 1779 bp nucleotides and encodes a protein of 593 amino acids.Both sequences possess the bHLH conserved domain which is a common feature of bHLH family protein.The expression of both the selected genes,at different flowering stages of H29 was also measured by Quantitative Real Time qRT-PCR.Which showed the highest expression of both genes at full blooming flower stage which indicates their possible involvement and important role during blooming especially with aspect to volatile organic compounds production and pigmentation in flowers.The phylogenetic analysis of these genes with different plants predicted them as MYC2 and bHLH13/JASMONATE ASSOCIATED MYC2-like2（JAM2）transcription factors both belonging to bHLH family.Overexpression of CpMYC2 in Arabidopsis（Arabidopsis thaliana）AtMYC2-2 Mutant（Salk₀83483）and Tobacco（Nicotiana tabaccum）genotype Petit Havana SR1 shows significant increment in floral volatile monoterpene especially Linalool in both plant types,while overexpression of CpbHLH13 in Arabidopsis thaliana ecotype Columbia-0（Col-0）and tobacco genotype Petit Havana SR1 significantly increases floral sesquiterpene ?-caryophyllene production in both types of transgenic plant which was measured by Gas Chromatography-Mass Spectroscopy（GC-MS）.Besides the ?-caryophyllene production,CpbHLH13 transformed tobacco plants showed the loss of anthocyanin in their inflorescence which may indicate the diversified role of the transcription factor CpbHLH13 as it shares 63% sequence similarity with Nelumbo nucifera,NnbHLH13/JAM2 and 42% identity with Arabidopsis AtbHLH13/JAM2 respectively.The TPS genes related to the production of monoterpenes TPS（At1g61680）and sesquiterpenes TPS（At5g23960 and At5g44630）also showed the higher expression in transgenic Arabidopsis thaliana mutant plants than the non-transferred wild type and mutant Arabidopsis plants.These results demonstrate that after transformation,both CpMYC2 and CpbHLH13 transcription factors from wintersweet are possibly involved in the positive regulation and biosynthesis of monoterpene（Linalool）and sesquiterpene（?-caryophyllene）when transferring in other plants as we found results from transgenic Arabidodpsis and tobacco plants.Methyle Jasmonic acid（MeJA）combine with Gibberellin（GA）has been proved to increase the expression of MYC2 transcription factor that enhances the TPS genes function,thus emit more volatiles from plants.The application in combination of Methyl Jasmonic Acid（MeJA）and Gibberellic acid（GA3）showed increment in the Linalool production in CpMYC2 overexpressed Arabidopsis plants along with more transcript level of TPS responsible for Linalool production.Thus we could suggest that CpMYC2 may be involves in Jasmonic acid（JA）signaling pathway to produce the terpenes in flowers and also involves in the anthocyanin producing mechanism as found in the loss of anthocyanin in transgenic tobacco plants.Our results draw the attention to initiate the deep study of these two wintersweet bHLH transcription factors involved in volatile organic compounds（VOC）production and anthocyanin regulation.As well as also provide a base to study the metabolism and molecular mechanism about the magnificent fragrance emits from the inflorescence of wintersweet.Moreover,it will not only open the new insights to understand floral terpene production but also contributes the great value in genetic engineering for the volatiles production in plants.In this way we can also use wintersweet as a valuable genomic material for genetic modification of floral scent in other flowering plants especially emitting low volatiles from their inflorescence.